Paenibacillus XP02 and application thereof in fermentation of poria cocos powder

Abstract

The invention relates to the technical field of microorganisms, in particular to paenibacillus XP02 and application of the paenibacillus XP02 in fermentation of poria cocos powder. The invention provides paenibacillus sp. XP02 with the capability of efficiently degrading insoluble poria cocos powder, and the paenibacillus sp. XP02 can be converted into metabolites such as soluble poria cocos oligosaccharide and the like. According to the application, paenibacillus XP02 is fermented in a fermentation culture medium containing 30mg / mL of poria cocos powder for 120 hours, so that the conversion rate of soluble oligosaccharide is 5%. The method provides a new thought for the fields of extraction and conversion of polysaccharide substances, and also provides a new efficient production way for obtaining the poria cocos oligosaccharide. Compared with a traditional process, the method has the advantages that the process is simplified, the cost is reduced, meanwhile, the utilization rate of components which are originally difficult to utilize in poria cocos is increased, and the method has important application value in the fields of biotransformation, food industry, medicine and health care and the like.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to Paenibacillus XP02 and its application in fermenting Poria cocos powder. Background Art

[0002] Poria, also known as Poria cocos and Pine and Cypress Jade, is the dried sclerotium of the Polyporaceae fungus Poria cocos (Wolfiporia cocos). Poria cocos is a traditional Chinese medicinal material for both medicine and food, which is included in the 2015 Chinese Pharmacopoeia Volume 1. It is sweet, light and flat in nature and flavor; it enters the heart, lung, spleen and kidney meridians; it has the effects of diuresis, invigorating the spleen and calming the mind; it is mainly used to treat edema, oliguria, phlegm and fluid, palpitations, spleen deficiency, poor appetite, loose stools, restlessness, palpitations and insomnia. Poria cocos is one of the components of the "Four Gentlemen and Eight Treasures" of traditional Chinese medicine. It has a wide range of uses and large amounts. There are hundreds of Chinese patent medicines produced using Poria cocos as raw materials. At the same time, it is also used in nutrition, health care and medicinal food.

[0003] Poria cocos mainly includes two types of chemical components: Pachyman (PM) and triterpenoids. PM is the most abundant component in Poria cocos, accounting for about 70%-90% of the dry weight of Poria cocos. PM can be divided into water-soluble polysaccharides and water-insoluble polysaccharides according to its solubility in water. Most of them are water-insoluble polysaccharides, and water-soluble polysaccharides only account for 0.5% of the total polysaccharides. Studies have shown that untreated water-insoluble PM has no biological activity and is difficult to use in the production of Chinese patent medicines and other pharmaceutical preparations. Therefore, traditional Chinese medicines often discard it as drug residues, which causes a large amount of waste and pollution of raw materials. In recent years, the solubilization modification of Poria cocos polysaccharides has mainly been carried out by introducing functional groups through chemical methods to modify its structure, such as carboxymethylation, sulfation, phosphorylation, etc. However, the chemical method has the disadvantages of high energy consumption and solvent residue. It is very necessary to explore biological processes as an alternative method for modifying the structure of polysaccharides. Biological modification mostly prepares oligosaccharides by enzymatic degradation of polysaccharides, for example, using hydrolases to directional remove glycosidic bonds to reduce the molecular weight and obtain water-soluble Poria cocos polysaccharides. At present, microbial fermentation is mostly used as a pretreatment method to improve the extraction rate of polysaccharides, and there are few reports on the direct degradation of polysaccharides by microbial fermentation. Microbial fermentation uses intact microbial cells as biocatalysts to catalyze the conversion of exogenous compounds, which can shorten the production cycle and reduce production costs. However, there are no reports on the fermentation of Poria cocos by Paenibacillus strains. Summary of the invention

[0004] In view of this, the present application screened a strain of Paenibacillus sp. XP02 that can efficiently degrade insoluble Poria cocos, and convert the insoluble Poria cocos powder substrate into soluble oligosaccharides, overcoming the shortcomings of the traditional method of obtaining Poria cocos powder, such as difficulty in solubility, low conversion rate, and environmental pollution, and has the advantages of simple conversion process and high application value.

[0005] The technical solution of this application is implemented as follows:

[0006] In the first aspect, the present application provides a Paenibacillus XP02, which is Paenibacillus sp. XP02, which has been deposited in the China Center for Type Culture Collection on November 12, 2024, with a deposit number of CCTCC NO: M 20242534, and a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] In a second aspect, the present application provides the use of the Paenibacillus XP02 in fermenting Poria powder, comprising: inoculating the Paenibacillus XP02 into a fermentation medium containing Poria powder for fermentation culture.

[0008] In some preferred embodiments, the fermentation medium is SM liquid medium or BHI liquid medium, and the content of Poria cocos powder in the fermentation medium is 10-40 mg / mL.

[0009] Furthermore, the application includes: inoculating Paenibacillus XP02 into a seed culture medium to prepare a seed solution, inoculating the seed solution into a fermentation culture medium containing Poria powder, and fermenting and culturing at 35-39° C. and 150-250 rpm.

[0010] In some preferred embodiments,

[0011] Each 200 mL of the SM liquid culture medium includes the following components: KCl 0.9 g, NaCl 0.9 g, NaHCO3 0.3 g, KH2PO4 0.1 g, K2HPO4 0.1 g, NH4Cl 0.06 g, MgSO4 0.04 g, alkaline trace elements 200 μL and acidic trace elements 200 μL; wherein,

[0012] The alkaline trace elements include Na2SeO3 0.1 mM, Na2WO4 0.1 mM, Na2MoO4 0.1 mM, and NaOH 10 mM;

[0013] The acidic trace elements include FeCl2 7.5 mM, H3BO4 1 mM, ZnCl2 0.5 mM, CuCl2 0.1 mM, MnCl2 0.5 mM, CoCl2 0.5 mM, NiCl2 0.1 mM, and HCl 50 mM.

[0014] The components of the seed culture medium include: each 200 mL of the seed culture medium includes the following components:

[0015] KCl 0.9g, NaCl 0.9g, NaHCO3 0.3 g, KH2PO4 0.1 g, K2HPO4 0.1 g, NH4Cl 0.06g, MgSO4 0.04 g, alkaline trace elements 200μL, acidic trace elements 200μL and Poria powder 4g;

[0016] The alkaline trace elements include Na2SeO3 0.1 mM, Na2WO4 0.1 mM, Na2MoO4 0.1 mM, and NaOH 10 mM;

[0017] The acidic trace elements include FeCl2 7.5 mM, H3BO4 1 mM, ZnCl2 0.5 mM, CuCl2 0.1 mM, MnCl2 0.5 mM, CoCl2 0.5 mM, NiCl2 0.1 mM, and HCl 50 mM.

[0018] Further, in some preferred embodiments, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 1-10% V / V.

[0019] In a third aspect, the present application provides a bacterial agent containing the Paenibacillus XP02.

[0020] Furthermore, the bacterial agent is a liquid bacterial agent, a solid bacterial agent or an immobilized bacterial agent.

[0021] In some preferred embodiments, the number of viable bacteria of the Bacillus XP02 in the bacterial agent is not less than 10 5 CFU / mL or 10 5 CFU / g.

[0022] The beneficial effects of this application are:

[0023] The Bacillus XP02 screened in this application can directly and efficiently degrade insoluble Poria powder and convert it into soluble metabolites such as Poria oligosaccharides, providing a new idea for the extraction and conversion of polysaccharides. In a fermentation medium containing 30 mg / mL Poria powder, after 120 hours of fermentation, Bacillus XP02 achieved efficient degradation of Poria powder and obtained a soluble Poria oligosaccharide conversion rate of up to 5%. This method provides a new and efficient production route for obtaining Poria oligosaccharides, significantly improving the conversion rate and metabolic efficiency. In addition, compared with traditional processes, the method of fermenting Poria powder with Bacillus XP02 can simplify the production process, effectively shorten the production cycle, and also reduce production costs. In addition, this method also helps to improve the utilization rate of difficult-to-use components in Poria, helps to reduce the generation of waste, and has positive significance for environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a transparent circle result diagram of the metabolism of Poria cocos strains 1-8 in the examples of this application;

[0026] Figure 2 This is a colony morphology diagram of Paenibacillus XP02 of the present application, wherein the lower right part is 4 times magnified;

[0027] Figure 3 This is a Gram staining microscopic image of Paenibacillus XP02 of the present application;

[0028] Figure 4 This is the phylogenetic tree of Paenibacillus XP02 of this application;

[0029] Figure 5 This is the test result of reducing sugar and total sugar in Poria powder fermented by Paenibacillus XP02 for 120 hours. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the following examples are only used to illustrate the present application and in no way limit the scope of protection of the present application. The experimental methods for which detailed conditions are not indicated in the following examples are usually carried out according to conventional conditions such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The experimental materials used in the examples of the present application can be obtained from commercial channels unless otherwise specified. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0031] The configuration of the culture medium in the examples of the present application is as follows:

[0032] SM+ Poria powder liquid culture medium (200mL): KCl 0.9g, NaCl 0.9g, NaHCO30.3g, KH2PO40.1g, K2HPO40.1g, NH4Cl 0.06g, MgSO40.04g, alkaline trace elements 200μL, acidic trace elements 200μL, Poria powder (purchased from Fenghe Biotechnology Company) 4g; Among them,

[0033] Alkaline trace elements: Na2SeO3 0.1 mM, Na2WO4 0.1 mM, Na2MoO4 0.1 mM, NaOH 10 mM;

[0034] Acidic trace elements: FeCl27.5 mM, H3BO41 mM, ZnCl20.5 mM, CuCl20.1 mM, MnCl20.5 mM, CoCl20.5mM, NiCl20.1 mM, HCl 50mM.

[0035] SM+Poria powder solid culture plate: Add 20g / L agar powder to the above SM+Poria powder liquid culture medium.

[0036] Brain heart infusion broth (BHI) was purchased from Qingdao Haibo Biotechnology Co., Ltd., product number: HB8297-1.

[0037] Seed culture medium (200 mL): KCl 0.9 g, NaCl 0.9 g, NaHCO3 0.3 g, KH2PO4 0.1 g, K2HPO4 0.1 g, NH4Cl 0.06 g, MgSO4 0.04 g, alkaline trace elements 200 μL, acidic trace elements 200 μL and Poria powder 4 g; wherein, the configuration of alkaline trace elements and acidic trace elements is the same as above.

[0038] Example 1 Isolation, purification and identification of Paenibacillus strain XP02

[0039] (1) Isolation and purification of strains

[0040] Fresh feces were sampled from 3 healthy volunteers (without digestive system diseases and without any antibiotic treatment within 3 months), and liquid screening medium (SM + Poria powder liquid medium) was added and cultured for 2 days. The fermentation broth was diluted with sterile PBS, and 100 μL of each gradient dilution was respectively taken and spread on SM + Poria powder solid culture plates, and cultured in a constant temperature incubator at 37°C until a single bacterium grew.

[0041] In the clean bench, pick single colonies of different colors and morphologies on the SM+Poria powder solid culture plate, inoculate them in BHI liquid culture medium for enrichment, and then streak them on BHI solid culture medium for gradual separation and purification until a single strain is obtained. Inoculate the single bacteria numbered 1-8 on the SM+Poria powder solid culture plate, culture at a constant temperature of 37℃ for 120h, and observe the transparent circle. Figure 1 As shown, strain No. 6 with a shorter transparent zone formation time and a larger transparent zone diameter was selected as the strain that can metabolize Poria powder.

[0042] (2) Identification of strains

[0043] Colony morphology identification: Take the bacterial solution of strain No. 6 above, plate it with glass beads on BHI solid medium, and observe the following after culturing at 37℃ for 24h: Figure 2 The colony morphology is circular, milky white or off-white, with a smooth and shiny surface and irregular edges.

[0044] Gram staining microscopic morphological identification: After Gram staining, observe the bacterial morphology under an optical microscope, such as Figure 3 As shown, the bacteria are blue and are Gram-positive bacteria. The cell morphology under the microscope is rod-shaped.

[0045] 16S rDNA sequencing identification: The 16S rDNA sequencing result of strain 6 is shown in SEQ ID NO.1, which was submitted to the NCBI database for homology comparison and a phylogenetic tree was drawn, as shown in Figure 3 shown.

[0046] Combined with the above test results, strain No. 6 was identified as Paenibacillus sp. XP02, which was deposited in the China Center for Type Culture Collection on November 12, 2024. The abbreviation of the preservation unit name is CCTCC, the preservation number is CCTCC NO: M 20242534, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0047] Example 2

[0048] The Bacillus strain XP02 on the BHI solid medium in step (2) of Example 1 was selected and inoculated into a BHI liquid medium with a single clone of bacteria. The culture was placed in a constant temperature shaker at 200 rpm for 12 hours to obtain a seed culture medium. The seed culture medium was then inoculated into a SM+Poria powder liquid culture medium at an inoculum amount of 2% (V / V). The culture was placed in a constant temperature shaker at 200 rpm for 120 hours, and samples were taken every 24 hours; the samples at each time point were centrifuged at high speed to obtain the supernatant, and after removing the insoluble matter and bacteria, the total sugar and reducing sugar were detected.

[0049] The detection method is as follows:

[0050] The total sugar was detected by sulfuric acid phenol method. The conditions included: take 150 μL of sample supernatant, add 100 μL of 6% phenol and mix thoroughly, then quickly add 500 μL of concentrated sulfuric acid on ice. After mixing and cooling, boil in water bath for 20 minutes, and after cooling, pipette 200 μL into 96-well plate and detect OD 490 . Establishment of standard curve: Prepare 1 mg / mL glucose solution and dilute to 700, 600, 500, 400, 300, 200, 100, 50, 0 μg / mL respectively, and determine the OD of each concentration of glucose according to the above method. 490 And draw a standard curve. Convert the total sugar concentration according to the sample absorbance value.

[0051] The reducing sugar was detected by DNS method. The conditions included: taking 220 μL of sample supernatant, adding 440 μL of DNS reagent and mixing thoroughly, boiling in water bath for 5 min, cooling and then pipetting 200 μL into a 96-well plate, and detecting OD 540 . Establishment of standard curve: Prepare 5 mg / mL glucose solution and dilute to 2.5, 2, 1.5, 1, 0.5, 0 mg / mL respectively, and determine the OD of each concentration of glucose according to the above method. 540 And draw a standard curve. Convert the reducing sugar concentration according to the sample absorbance value.

[0052] Test results such as Figure 5As shown in the figure, during the fermentation of Paenibacillus strain XP02, the change of total sugar content in the fermentation broth showed an initial slight decline, followed by a significant upward trend; the change of reducing sugar content also showed the same trend. In the initial stage of fermentation, the content of total sugar and reducing sugar both showed a slight decline. This phenomenon can be attributed to the rapid consumption of sugars by strain XP02. The strain began to use these sugars as energy sources and substrates for biosynthesis at the beginning of fermentation. The slight reduction in sugar content is a direct result of the strain adapting to the new environment and starting metabolic activities. As the fermentation progressed, the content of total sugar and reducing sugar began to increase significantly from about 24 hours. This change indicates that strain XP02 has successfully initiated a specific conversion mechanism for Poria powder. As the only carbon source, Poria powder is decomposed into a variety of metabolites under the action of the strain, including water-soluble Poria oligosaccharides and some soluble reducing sugars. The test results reveal the application value of Paenibacillus strain XP02 in the efficient bioconversion of Poria powder.

[0053] Example 3

[0054] The Bacillus strain XP02 on the BHI solid medium in step (2) of Example 1 was selected and inoculated into a BHI liquid medium with a single clone of bacteria. The mixture was placed in a constant temperature shaker at 200 rpm and cultured for 12 hours to obtain a seed culture medium. The seed culture medium was then inoculated into a SM+ Poria powder liquid culture medium at an inoculum rate of 2% (V / V). The mixture was placed in a constant temperature shaker at 200 rpm and cultured for 120 hours. After sampling, the sample was centrifuged at high speed to obtain the supernatant. After removing the insoluble matter and bacteria, the total sugar and reducing sugar were detected according to the detection method in Example 2. The total sugar conversion rate and the reducing sugar conversion rate were respectively obtained by the following calculation method:

[0055] Total sugar conversion rate = total sugar concentration obtained by fermentation / original concentration of Poria powder added * 100%

[0056] Reducing sugar conversion rate = reducing sugar concentration obtained by fermentation / original concentration of Poria powder added * 100%

[0057] The test results showed that after 120h of fermentation in SM+Poria powder liquid medium, the conversion rates of total sugar and reducing sugar of Paenibacillus strain XP02 were 5% and 2.2%, respectively. Therefore, in the fermentation medium containing 30mg / mL Poria powder, after 120h of fermentation, Paenibacillus XP02 achieved efficient degradation of Poria powder and obtained a soluble Poria oligosaccharide conversion rate of up to 5%. This method provides a new and efficient production route for obtaining Poria oligosaccharides, significantly improving the conversion rate and metabolic efficiency.

[0058] The above description is only a preferred implementation mode of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Paenibacillus XP02, characterized in that The Bacillus XP02 is Paenibacillus sp. XP02, which was deposited in the China Center for Type Culture Collection on November 12, 2024, with a deposit number of CCTCC NO: M 20242534, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The use of Paenibacillus XP02 according to claim 1 in fermenting Poria powder, characterized in that: include: The Paenibacillus XP02 is inoculated into a fermentation medium containing Poria powder for fermentation culture.

3. The use according to claim 2, characterized in that: The fermentation medium is SM liquid medium or BHI liquid medium, and the content of Poria cocos powder in the fermentation medium is 10-40 mg / mL.

4. The use according to claim 2 or 3, characterized in that: include: The Paenibacillus XP02 was inoculated into a seed culture medium to prepare a seed solution, and the seed solution was inoculated into a fermentation culture medium containing Poria powder, and fermented and cultured at 35-39° C. and 150-250 rpm.

5. The use according to claim 3, characterized in that: Each 200 mL of the SM liquid culture medium includes the following components: KCl 0.9 g, NaCl 0.9 g, NaHCO3 0.3 g, KH2PO4 0.1 g, K2HPO4 0.1 g, NH4Cl 0.06 g, MgSO4 0.04 g, alkaline trace elements 200 μL and acidic trace elements 200 μL; wherein, The alkaline trace elements include Na2SeO3 0.1 mM, Na2WO4 0.1 mM, Na2MoO4 0.1 mM, and NaOH 10 mM; The acidic trace elements include FeCl2 7.5 mM, H3BO4 1 mM, ZnCl2 0.5 mM, CuCl2 0.1 mM, MnCl2 0.5 mM, CoCl2 0.5 mM, NiCl2 0.1 mM, and HCl 50 mM.

6. The use according to claim 4, characterized in that: Each 200 mL of the seed culture medium includes the following components: KCl 0.9g, NaCl 0.9g, NaHCO3 0.3 g, KH2PO4 0.1 g, K2HPO4 0.1 g, NH4Cl 0.06g, MgSO4 0.04g, alkaline trace elements 200μL, acidic trace elements 200μL and Poria powder 4g; among them, The alkaline trace elements include Na2SeO3 0.1 mM, Na2WO4 0.1 mM, Na2MoO4 0.1 mM, and NaOH 10 mM; The acidic trace elements include FeCl2 7.5 mM, H3BO4 1 mM, ZnCl2 0.5 mM, CuCl2 0.1 mM, MnCl2 0.5 mM, CoCl2 0.5 mM, NiCl2 0.1 mM, and HCl 50 mM.

7. The use according to claim 4, characterized in that: The seed liquid is inoculated into the fermentation medium at an inoculum amount of 1-10% V / V.

8. A bacterial agent, characterized in that Containing the Paenibacillus XP02 according to claim 1.

9. The bacterial agent according to claim 8, characterized in that The bacterial agent is a liquid bacterial agent, a solid bacterial agent or an immobilized bacterial agent.

10. The bacterial agent according to claim 8 or 9, characterized in that: The viable count of the Bacillus XP02 is not less than 10 5 CFU / mL or 10 5 CFU / g.

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